ABSTRACT
DNA dumbbells are stable, short segments of double-stranded DNA with closed nucleotide loops on each end, conferring
resistance to exonucleases. Dumbbells may be designed to interact with
transcription factors in a sequence-specific manner. The internal based paired sequence of DNA dumbbells in
this study contains the X-box, a positive regulatory motif found in all MHC class II DRA promoters.
In electrophoretic mobility shift assays (EMSAs), dumbbells and other oligonucleotides (`decoys') with the core X-box sequence were found to compete with the native strand for binding to X-box binding proteins (including RFX1). However, only the X-box dumbbell was capable of forming detectable complexes with
such proteins using EMSA. In a model cell system, dumbbells were tested for
their ability to block RFX1VP16 activation of a plasmid containing multiple
repeats of the X-box linked to the
CAT
gene. While it appeared that dumbbells could block this activation, the effect
was non-specific. This and further evidence suggests an inhibition of transcription, most likely via an interaction with the general transcriptional machinery.
Selective regulation of gene expression is a direct way to treat major diseases
such as cancer, viral infections and various genetic disorders. While there are
numerous reports on strategies targeting mRNA via antisense oligonucleotides (
1
) and DNA via triplex formation (
2
), targeting the proteins which regulate expression of a specific gene is a
relatively novel approach. These `gene regulating' proteins are transcription
factors (TFs) which bind to DNA in a sequence-specific manner. There have been several reported cases of utilizing
sequence-specific DNA decoys for selective sequestering of TFs, rendering them
unavailable to bind to their target DNA on the gene (
3
-
5
). The final result is a blockade of transcriptional activation.
Several different forms of double-stranded DNA have been utilized as decoys for TFs, including unmodified
oligonucleotide duplexes (
6
), [alpha][beta]-anomeric (chirally modified) oligonucleotides (
7
), phosphorothioate oligonucleotide duplexes (
3
) and dumbbell DNA (
5
). Use of phosphorothioate oligonucleotide duplexes may overcome the inherent
disadvantage of nuclease susceptibility that is typical of unmodified
oligonucleotides. However, recent studies have shown that double-stranded phosphorothioate oligonucleotides may exhibit sequence-independent effects due to non-specific protein binding (
8
). Chirally modified oligonucleotides are also nuclease resistant, but they require special synthesis techniques. It is also unclear which chiral form(s) will be recognized by a given TF, if
at all.
Dumbbell DNA, on the other hand, has increased stability to exonucleases (
9
), is easy to synthesize, cannot undergo strand separation and has a non-toxic unmodified backbone which resembles natural DNA. These
characteristics make dumbbell DNA an excellent candidate for TF binding and
competition studies. While dumbbell DNA has been extensively studied as a
physical model for various DNA conformations (
10
-
12
), little is known about its biological relevance, i.e. if dumbbell DNA will
even be recognized by its target TF. On a rudimentary level, if dumbbell DNA
has a specified DNA recognition sequence, it should. However, compared to a
long, typical duplex segment of DNA, dumbbell DNA might adopt a different, abiotic conformation. Additionally, the TF itself may require flanking sequences or other factors for binding in addition to the specific DNA recognition sequence.
Dumbbells with a loop composition of all thymidines (Ts) and a loop size of 4 nt
were chosen for our initial studies based on several DNA hairpin studies.
Blommers
et al
. (
13
) showed that a particular DNA hairpin was maximally stable when the loop was
comprised of 4 (or 5) nt. Additionally, Senior (
14
) found that for their specific hairpin, homonucleotide loops were the most
stable. Additional experiments utilized all A loop dumbbells and highly stable
CTTG loop dumbbells.
The internal duplex sequence of DNA dumbbells and other double-stranded oligonucleotides were designed to mimic the X-box of the DRA promoter, essential in regulating expression of the
Major Histocompatibility Complex (MHC) class II gene. The X-box is a positive regulatory motif conserved in all MHC class II
promoters. Several TFs appear to target that sequence, including the RFX (Regulatory Factor X) family of TFs (for a review see
15
).
In
in vitro
binding assays, the X-box dumbbell was found to interact with at least one of the RFX family
members, RFX1. To verify these results
in vivo
, a reporter gene assay was devised to elucidate whether dumbbells could block
RFX1 activity. A plasmid containing the cDNA of RFX1 fused to the transcriptional activation domain of adenovirus VP16 was constructed (pRFX1VP16). The product
of this plasmid can bind to and activate p4XBCAT, which contains four repeats
of the X-box linked to the
CAT
gene. The initial results of this assay showed that oligonucleotides with the
core X-box sequence were able to block RFX1 activation. Surprisingly, ligated
dumbbells were no more active than unligated oligonucleotides. However, control
oligonucleotides were also active and transcription of unrelated reporter gene
systems was also reduced. Oligonucleotides had no effect on transfection
efficiency of plasmids based on a PCR assay. However, an RNase protection assay
indicated that active oligonucleotides decreased mRNA levels, suggesting an
effect on polymerase II or its associated transcriptional machinery.
Oligonnucleotides were obtained from either Keystone Laboratories (Menlo Park, CA) or Oligos Etc. (Wilsonville, OR). Control oligonucleotides
contain irrelevant sequences. Oligonucleotide sequences and nomenclature are
listed in Table
1
. `Core X-box oligonucleotides' refers to all oligonucleotides with the X-box internal sequence (these include all oligonucleotides listed in
Table
1
except for the `ctrl' oligonucleotides).
An intramolecularly annealing (i.e. self-complementary) oligonucleotide was phosphorylated (with T4 polynucleotide kinase) then ligated
(with T4 DNA ligase) to form dumbbell DNA as described elsewhere (
16
). For radiolabeled oligonucleotides/dumbbells (
32
P phosphorylation) in a 10 [mu]l volume, 10 pmol oligonucleotide was incubated with 1 U T4 polynucleotide kinase (US Biochemical), 20 [mu]Ci [[gamma]-
32
P]ATP (Amersham), 50 mM Tris-HCl, pH 7.6, 10 mM MgCl
2
and 10 mM 2-mercaptoethanol at 37oC for 1 h. Ligated refers to dumbbell products and unligated (unl)
refers to the starting reactant oligonucleotide. For ligation of
32
P-labeled oligonucleotides, 5 pmol
32
P-labeled oligonucleotide was ligated in a 10 [mu]l volume with 1 U T4 DNA ligase, 5% polyethylene glycol, 66 mM Tris-HCl, pH 7.6, 6.6 mM MgCl
2
, 10 mM DTT and 66 [mu]M ATP at 16oC for 48 h.
Dumbbells were purified on 12% denaturing (8.5 M urea) polyacrylamide gels (
17
). The ligation reaction mixtures were visualized by UV shadowing and products (dumbbells) were isolated by slicing and removing
the appropriate bands. Products were eluted from gel slices for 30 min using
the BioRad Model 422 electroeluter, ethanol precipitated and purified on NAP-5 (Sephadex G-25) columns (Pharmacia LKB, Uppsala, Sweden) using 1 mM Tris-HCl, 0.1 mM EDTA, pH 7.6. Radiolabeled
oligonucleotides/dumbbells were similarly gel purified (but not electroeluted).
Gel slices from these reactions were instead eluted overnight with 100 [mu]l TE (10 mM Tris-HCl, 1 mM ETDA, pH 7.6) at room temperature. Unligated
oligonucleotides were purified by HPLC by the manufacturer and used directly.
Proof of ligation experiments have been described elsewhere (
16
) and are summarized as follows. The product (dumbbell DNA) was subjected to
three enzymes: the Klenow fragment of
Escherichia coli
, S1 nuclease and shrimp alkaline phosphatase. In all three cases, dumbbell DNA
was more resistant to degradation compared with its corresponding unligated sequence. The restriction enzyme
Mae
I was used to show that the dumbbell DNA formed a cleavable, recognizable
duplex.
Class II-expressing Raji cells (an EBV-positive human Burkitt's lymphoma B cell line) were grown in RPMI
1640 medium with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 [mu]g/ml) and L-glutamine (292 [mu]g/ml). Cos7 cells, a SV40-transformed monkey kidney cell line, were grown in
Dulbecco's modified Eagle's medium (with 4.5 g/l glucose) and FBS and
antibiotics as above. Nuclear extracts were prepared from Raji cells as
described previously (
18
), except that the supernatant was not diluted in the last step. [alpha]RFX1 (anti-RFX1 antibody), which recognizes the N-terminus of recombinant RFX1, was prepared as previously described (
19
).
Competition assay.
Nuclear extract containing RFX1 was incubated with radiolabeled DRAX and various unlabeled competitors. The X dumbbell, unl X, ctrl dumbbell 1, unl ctrl 1, ds X, ds S-X or ns 10 was used as competitor at 150-fold excess over radiolabeled DRAX. Specifically, the binding assay
at 0oC included 10 [mu]g nuclear extract and 10 pmol unlabeled competitor in the binding
buffer described by Reith (
20
) with 1-2 [mu]g poly(dI[middot]dC)[middot]poly(dI[middot]dC) incubated for 10 min, followed by the addition of
32
P-labeled DRAX probe (0.066 pmol or 12 000 c.p.m.) for an additional 20 min. For this and the following EMSA gels, samples
were loaded onto 5% native polyacrylamide gels and run with recirculating 0.25* TBE buffer for 2-3 h at 10 mA and 150 V at room temperature. After drying, gels were exposed to X-ray film for 1-3 days at -70oC.
RFX1 supershift with antibody.
Nuclear extract was incubated with either 2 [mu]l [alpha]RFX1 or 10 pmol unlabeled competitor oligonucleotide (DRAX or X-box dumbbell) under the conditions described above, with the addition of 50 ng
E.coli
DNA and 0.1% Nonidet P-40 at the start of incubation. After 10 min, radiolabeled DRAX was added
and the mixture was incubated for an additional 20 min.
Nuclear extract was incubated with radiolabeled oligonucleotides/ dumbbells (0.066 pmol or 15 000 c.p.m.) for 20 min at 0oC under the conditions mentioned for the competition assay.
RFX1 activation of 4XBCAT.
Aliquots of 1 * 10
5
Cos7 cells were plated in 6-well plates the day before transfection. Plasmids pRFX1VP16 (2 [mu]g) and p4XBCAT (1 [mu]g) plus 50 nM oligonucleotide were co-transfected with lipofectin (Gibco BRL, Gaithersberg, MD)
using the manufacturer's protocol. The concentration of the oligonucleotide in
the transfecting medium was four times that of the final oligonucleotide
concentration. In other words, for an oligonucleotide with a final
concentration of 50 nM, the concentration in the transfecting medium was 200
nM. Transfections were performed at least twice in triplicate. For RNase
protection assays, large scale transfections of 5 * 10
5
Cos7 cells in 100 mm plates (2.4 [mu]g pRFX1VP16 and 1.6 [mu]g p4XBCAT, 50 nM oligonucleotides) were performed.
Control transfections.
In separate transfections, 0.5 [mu]g CMV promoter-driven plasmid pCRtm3-CAT (Invitrogen, San Diego, CA) was used as an expression
control plasmid. These control transfections were performed at least twice in
duplicate. Another control utilized 2 [mu]g pSV[beta]gal (Promega, Madison, WI), transfected into 2 * 10
5
cells on 60 mm plates (performed twice in triplicate). Both control
transfections included various oligonucleotides at 50 nM and were performed on
Cos7 cells.
Plasmid constructs.
pRFX1VP16 was constructed by first amplifying the RFX1 cDNA (a gift of B.Mach)
with primers that introduced a
Hin
dIII site after the final amino acid codon. This cDNA was subcloned into
pSVSPORT1 (Life Technologies, Grand Island, NY) and the cDNA coding for the
activation domain of VP16 from herpes simplex virus (amino acids 400-479) was ligated into the introduced
Hin
dIII and
Sal
I sites of the plasmid. The plasmid p4XBCAT contains four X-box repeats and has been described previously (
21
).
CAT assays.
Cells were harvested for CAT assays ~48 h after electroporation. CAT assays were performed on cell lysates as
described previously (
22
,
23
). Protein content (A
595
), determined using the Bradford assay (using BioRad Protein Assay Reagent), was
used to normalize CAT activity. In some cases cell counts were also taken to
determine viability.
[beta]
-Galactosidase assays.
Cells were stained
in situ
for [beta]-galactosidase activity and visually inspected for blue cells. A
qualitative result was obtained by removing the cells from the plates with
trypsin and counting blue cells.
DNA was extracted from cells transfected with 2 [mu]g pRFX1VP16 and 1 [mu]g p4XBCAT with or without oligonucleotide using a modified Hirt assay (
24
) with an incubation time and temperature of 1 h and 50oC respectively. Each PCR reaction in a total volume of 100 [mu]l contained 10 [mu]l template DNA, 0.2 mM dNTPs, 100 pmol primers V1/V2 (for pRFX1VP16; V1, GCC ACC ATG GCA TCG ACG GCC CCC
CCG ACC GAT; V2, GTC GAC CCC ACC GTC CTC GTC AAT TCC), 1 U Taq DNA polymerase
(Boehringer Mannheim), 10 mM Tris-HCl, 50 mM KCl, pH 8.3, and 2.5 mM MgCl
2
. All samples (including controls) were subjected to 25 cycles of amplification
(30 s denaturing at 94oC, 30 s annealing at 60oC, 60 s extension at 74oC) and were loaded onto a 1.5% agarose gel (with ethidium bromide) and photographed under UV illumination for analysis.
Cells from large scale transfections were harvested as pooled duplicates, 48 h post-transfection. Cytoplasmic RNA was isolated from these cells using the method of Kao
et al
. (
25
) for use in RNase protection assays. The RNA probe was prepared by linearizing pCRtm3-CAT with
Pvu
II. This created three fragments, one of which was
in vitro
transcribed by T7 polymerase using the MAXIscripttm system (Ambion, Austin, TX) to generate a
32
P-labeled fragment of 173 nt. This probe was complementary to a 111 nt
region of the CAT transcript. In addition, a
32
P-labeled 18S rRNA internal standard probe (116 nt transcript, 80 nt of
which are complementary to rRNA from all vertebrates) was prepared using
linearized pT7 RNA 18S (Ambion). Protection assays were performed using the
Guardian RNase protection assay kit (Clontech, Palo Alto, CA) with
hybridization at 42oC overnight. CAT RNA and 18S RNA were probed separately. Samples were
heated at 95oC for 5 min before loading onto a 6% denaturing (8 M urea) acrylamide gel
and electrophoresed in TBE buffer for 1.5-2 h at 500 V. After drying, gels were exposed to X-ray film overnight at -70oC.
In order to determine which oligonucleotides/dumbbells interact with X-box binding proteins, EMSAs were performed. B cell nuclear extracts were
first incubated with a radiolabeled 26 bp X-box probe (DRAX) with or without competitor oligonucleotides/ dumbbells. The unlabeled competitors, X dumbbell, unl X, ds X and the ds S-X (Fig.
1
, lanes 3, 4, 7 and 8 respectively) successfully competed with DRAX for binding
to X-box binding proteins. As little as a 30-fold excess of unl X was able to compete with DRAX (data not shown).
Oligonucleotides with irrelevant sequences were unable to compete for the
formation of shifted complexes (Fig.
1
, lanes 5, 6 and 9).
To ascertain if RFX1 interacts with dumbbell DNA, a supershift assay was
utilized. Inclusion of [alpha]RFX1 in the EMSA binding reaction resulted in a decrease in the
electrophoretic mobility of the complex, indicating that it contained RFX1 (Fig.
2
B, lane 1, a). The X dumbbell was able to compete for and (weakly) bind to the RFX1-containing complex and the lower unidentified complex as well (Fig.
2
b, lane 4, b). This lower band is most likely another member of the RFX family (
27
,
28
).
Since core X-box oligonucleotides/dumbbells were able to interact with RFX1
in vitro
, the next step was to determine if these oligonucleotides/dumbbells could specifically inhibit transcriptional activation by RFX1
in vivo
. The plasmid pRFX1VP16 expresses a protein consisting of RFX1 fused to the
strong VP16 transcriptional activating domain of herpes simplex virus. This
fusion protein is a potent transcriptional activator dependent only on RFX1
binding to its target sequence. The p4XBCAT plasmid contains multiple repeats
of the X-box, the target for RFX1, which allows for strong transactivation.
Dumbbells, unligated oligonucleotides, double-stranded oligonucleotides and double-stranded phosphorothioate oligonucleotides were tested for their
ability to block RFX1VP16 activation of p4XBCAT. Initial studies indicated that optimal (e.g. maximal effect with no toxicity) oligonucleotide concentrations in Cos7 cells were in the range 50-100 nM (data not shown). The phosphorothioate oligonucleotide (ds S-X) at 50 nM always had higher or similar cell counts (viability) compared with no oligonucleotide added (data not
shown).
Since the ligated dumbbell (X dumbbell) was the only oligonucleotide capable of
forming a detectable complex with RFX1 (under the conditions of these EMSAs),
it was anticipated that they would be the most potent inhibitors of RFX1VP16
activation of 4XBCAT. Clusel
et al.
showed that ligated (but not unligated) dumbbells specific for the
transcription factor HNF-1 were able to block HNF-1 activation of a CAT reporter plasmid (
5
). In our system, however, it was found that ligated dumbbells were no more
active than unligated in blocking RFX1VP16 activation of p4XBCAT (data not
shown). Thus, the activity of oligonucleotides in this system is not dependent
on a ligated, double-stranded structure.
As seen in Figure
3
, core X-box oligonucleotides, including unl X and ds S-X, were effective in down-regulating CAT activity. ds X was much less active, presumably
due to degradation by exonucleases. A hairpin X-box (loop on one end only) was of intermediate activity. Cell viabilities
were relatively stable among all samples. Significantly, control (irrelevant)
sequence unligated oligonucleotides (unl ctrl 2 and unl ctrl 3) inhibited
RFX1VP16 activation of 4XBCAT. The activity of these irrelevant oligonucleotides suggested a non-specific effect of unligated oligonucleotides in general on the function
of RFX1VP16. Another double-stranded phosphorothioate (with the same internal sequence as unl ctrl 3)
was also able to decrease RFX1VP16 activation of 4XBCAT to the same extent as
ds S-X (data not shown).
As control experiments, some of the core X-box oligonucleotides at 50 nM were tested for their effect on other
reporter gene systems, including CMVCAT and SV[beta]gal, neither of which were expected to be specifically regulated by any of
the core X-box oligonucleotides. CMVCAT transfection results are shown in Figure
4
. Reduction of reporter gene readout by various oligonucleotides in this system
followed the same trend as that of the RFXVP16/4XBCAT system.
The non-specific inhibition of transcription may have been due to the
oligonucleotides/dumbbells ability to reduce transfection efficiency. To
address this issue, Cos7 cells transfected with plasmids in the presence or
absence of oligonucleotide were harvested, lysed and the plasmid DNA subjected
to PCR amplification. The amount of DNA in each sample is proportional to the
band intensity when run on a gel if low cycle number (<25 cycles) PCR is used (
31
), i.e. in the linear amplification range. Two of the most active
oligonucleotides (ds S-X and unl CTTG-X) were tested. PCR analysis of transfected samples showed that the
amounts of amplification product of all transfections were similar (Fig.
5
, lanes 2-4), indicating that the oligonucleotides had no effect on transfection efficiency. Negative control lanes included mock-transfected cells (Fig.
5
, lane 1) and a water blank (Fig.
5
, lane 6). In addition to transfected pRFX1VP16, another positive control included 2 ng pRFX1VP16 as template (Fig.
5
, lane 5).
To determine if active oligonucleotides/dumbbells were effecting general
transcription of the reporter plasmid, mRNA transcripts were measured using an
RNase protection assay. Transfection of plasmids/oligonucleotides into Cos7
cells was performed as described and RNA was extracted. RNase assays revealed a
decrease in CAT mRNA (transcribed by polymerase II) upon the addition of 50 nM
core X-box oligonucleotides, (Fig.
6
, top panel; compare lane 1, no oligonucleotide added, to lanes 2-5). The ds S-X oligonucleotide (Fig.
6
, top panel, lane 2) yielded the largest decrease in mRNA levels. The protected
band(s) is the predicted size (111 bp). In contrast, levels of 18S RNA
(transcribed by polymerase I) were not altered by the addition of
oligonucleotides (Fig.
6
, bottom panel, lanes 2-5; CAT RNA and 18S RNA were probed separately). In addition, levels of
total RNA were not decreased by active oligonucleotides using a total RNA accumulation assay (data not shown).
In EMSAs using Raji nuclear extracts and a labeled X-box oligonucleotide probe, competitor oligonucleotides with the core X-box sequence were able to compete efficiently for complex formation.
However, in direct binding assays, only the X dumbbell was able to form a
detectable complex with an X-box protein identified as RFX1 under these conditions. Unligated
irrelevant sequence oligonucleotides did not compete
in vitro
, but had activity in three different
in vivo
reporter gene assays. This suggested that the oligonucleotides inhibited
transcription in a manner not predicted, i.e. not by competition with the
reporter plasmid target for RFX1 binding. The PCR-based assay indicated that the oligonucleotides were not directly
interfering with plasmid transfection. RNase protection assay analysis
indicated that the oligonucleotides were inhibiting RNA polymerase II
transcription in general.
Toxicity or cell viability is an important factor that should be considered when
interpreting results. In the typical reporter gene assays used for assessing
oligonucleotide activity, cell death can inadvertently contribute to
`activity', since activity is commonly measured by decreases in reporter gene
protein. While others have reported the sequence-specific blockage of transcription factors with oligonucleotide doses
ranging from 10 nM to 7.5 [mu]M, no mention of cell viability/toxicity is made in some of the studies (
3
,
5
). Some of our earlier studies indicated that certain oligonucleotides were more toxic than others, usually when in the micromolar range (data
not shown). There have been several reports of toxicity of phosphorothioates (
32
,
33
), however, in our studies, the ds S-X phosphorothioate (and another phosphorothioate with the same internal
sequence as unl ctrl 3) was never toxic (as measured by cell viability) at the
concentrations we tested (up to 5 [mu]M).
Factoring out toxicity, in our reporter gene assays all core X-box oligonucleotides were able to block RFX1VP16 activation of 4XBCAT.
Interestingly, activity correlated with predicted oligonucleotide stability
(i.e. the more stable the oligonucleotide, the more potent it was).
Phosphorothioates would be expected to be the most stable due to their nuclease
resistance in cells (
34
). Chu (
9
) reported the following order of stability (in human sera): dumbbells >
hairpins > double strands > single strands, due to active single-strand endonucleases and less rapidly acting double-strand exonucleases. Thermodynamically, among the unligated
dumbbells the expected order of stability is CTTG loop > T loop > A loop, based
on DNA hairpin studies (
11
). Therefore, in terms of resistance to exonucleases and thermodynamic
stability, the expected order of overall stability (and coincidently, the order
of activity) for our oligonucleotides was ds S-X > unl CTTG-X > unl X > unl A-X > hairpin X > ds X. The melting temperature of unl X under
highly denaturing conditions was 42.5oC, therefore, under physiological conditions unligated dumbbells would be
expected to exist as stable duplex molecules. Even with a nick in the dumbbell
(i.e. the unligated state), endonuclease degradation in this cell line could be
minimal, allowing the oligonucleotides ample time to exert their effects. The
stability of unligated dumbbells (and phosphorothioate oligonucleotides) may
contribute to their apparent activity.
In summary, factors that may influence the success of DNA decoys or dumbbells
include: the dose and toxicity profile of the oligonucleotide; the cell line;
the affinity and specificity of the transcription factor for the decoy
in vivo
; the affinity of the oligonucleotide toward transcriptional machinery; other non-specific effects. The study of DNA dumbbells presents a unique opportunity to test
the effects of the addition of short, unmodified or `naturally occurring'
phosphodiester oligonucleotide duplexes to cell systems. These dumbbells may have non-sequence-specific effects on the intracellular milieu, including inhibition of RNA
polymerase II transcription. Hence dumbbells may prove to be useful tools for
studies involving polymerase II transcription and gene expression.
Interestingly, unligated dumbbells (referred to as `nicked' dumbbells) with
phosphorothioate substitutions in the loops were recently found to have anti-HIV activity in a human T cell line (
35
). Unligated (nicked) dumbbells represent a new class of biologically active
oligonucleotides with activities yet to be discovered.
C.S.Lim was supported in part by a NIH Pharmaceutical Chemistry Training Grant.
The authors would like to thank Yoko S.Haga for
T
m
data and Thomas E.Cheatham for helpful discussions.
*To whom correspondence should be addressed at present address: Laboratory of
Receptor Biology and Gene Expression, National Cancer Institute, National
Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA. Tel: +1 301
496 7443; Fax: +1 301 496 4951; Email: limc@dce41.nci.nih.gov
Table 1
REFERENCES
Return